The Use of Covalently Closed Circular DNA to Investigate Properties of Bleomycin and Its Analogs

Abstract

The mechanism of action for bleomycin appears to involve production of both single- and double-strand DNA breakage^(1-3). A variety of methods have been used to investigate this phenomenon. Several investigators have used covalently closed circular DNA (CCC DNA) because of its unique properties in determining conformational alterations^(4-7). Bleomycin-induced single-strand breakage in superhelical eee DNA (form I) produces a relaxed open circular conformation (form II) and renders the DNA susceptible to alkali or heat denaturation. The product of this denaturation (single-strand DNA) can be detected by velocity sedimentation in alkaline sucrose gradients or another method depending on the loss of DNA intercalative binding by the fluorescent dye, ethidium bromide. Since the quantum yield of fluorescence by ethidium bromide is enhanced upon intercalative binding to double-stranded DNA⁽⁸⁾ a decreasein fluorescenceresults upon alkaline denaturation of broken eee DNA. This provides the basis for a fluorescent assay of DNAbreakage (Fig. I). Bleomycin-induced DNAfragmentation can also be detected using electrophoresis of eeeDNA. This is because the products of broken superhelicaleee DNA(form I), relaxed circular DNA(form II), and linear duplex DNA (form III) are electrophoretically distinguishable under appropriate conditions (Fig. 2)⁽⁹⁾. Electrophoresis also may be used to quantitate the double-strand breakage by bleomycin since production of linear duplex DNA(form III) requires a double-strand break.